Method of preventing mycoplasma infection

ABSTRACT

The effective ingredient in the inventive medicament against infection with mycoplasma is tea, e.g., black tea, or a tea polyphenol as a constituent of tea including epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin, (+) catechin and the isomer thereof, free theaflavin, theaflavin monogallates A and B and theaflavin digallate.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel preventive medicament againstinfection with mycoplasma or, more particularly, to a preventivemedicament against infection with mycoplasma having a mycoplasma-killingactivity so as to exhibit an effect for inhibiting infection withmycoplasma.

2. Background Information

Mycoplasma is a unique microorganism which, different from bacteria, isdevoid of cell walls to exhibit polymorphism and requires sterols andthe like. In the microbiological classification it is intermediatelypositioned between bacteria and viruses. Some of mycoplasmas are knownto act as a pathogenic microorganism against human beings including thepneumonic mycoplasma to cause pneumonia, ureaplasma to causenon-gonorrheal urethritis and the like. It is also reported that, inaddition to these infectious diseases in the respiratory organs andurinogenital organs, mycoplasmas may have some relevancy to theetiological factors to cause various diseases concomitantly occurring asa complication of the above mentioned diseases such as lesions in thecentral nerval system typically exemplified by lesions in the cerebralnerves, chronic articular rheumatism, acute salpingitis, acute pelvicdiseases and the like.

Despite the enormous volume of the investigational reports and the verylong history of studies on mycoplasmas, no conclusive results have yetbeen established of the pathogenicity of mycoplasmas so that knownchemotherapeutic medicaments are very limited including only macrolideantibiotics and tetracycline for the time being. Accordingly, it iseagerly desired to develop a novel anti-mycoplasma medicament which canbe administered to patients without undesirable side effects to thehuman body.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a novel medicamentagainst infection with mycoplasma as mentioned above. The inventors havecarried out extensive investigations of natural products to discover asubstance capable of exhibiting the desired effect without the problemsusually encountered by using chemically synthesized compounds.

Thus, the medicament of the present invention against infection withmycoplasma comprises tea as the medicinally effective ingredient.

Further, the medicament of the invention comprises polyphenol compoundsin tea as the effective ingredient. The polyphenol compound in tea asthe effective ingredient in the inventive medicament is selected fromthe group consisting of epigallocatechin gallate, epicatechin gallate,epigallocatechin, epicatechin, (+)catechin and the isomer thereof, freetheaflavin, theaflavin monogallate A, theaflavin monogallate B andtheaflavin digallate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a novel preventive medicament againstinfection with mycoplasma, of which tea is the effective ingredient.

The tea polyphenol compounds as the principal effective ingredients inthe inventive medicament against infection with mycoplasma include thetea catechin compounds represented by the general formula (I) givenbelow and the theaflavin compounds represented by the general formula(II) given below: ##STR1## in which R₁ is a hydrogen atom or a hydroxygroup and R₂ is a hydrogen atom or a 3,4,5-trihydroxy benzoyl group; and##STR2## in which R₃ and R₄ are, each independently from the other, ahydrogen atom or a 3,4,5-trihydroxy benzoyl group.

Particular examples of the tea catechin compounds represented by thegeneral formula (I) include:

(-)epicatechin, which is a compound of the formula (I) with R₁ =H and R₂=H; (-)epigallocatechin, which is a compound of the formula (I) with R₁=OH and R₂ =H; (-)epicatechin gallate, which is a compound of theformula (I) with R₁ =H and R₂₌ 3,4,5-trihydroxy benzoyl group; and(-)epigallocatechin gallate, which is a compound of the formula (I) withR₁ =OH and R₂₌ 3,4,5-trihydroxy benzoyl group. Particular examples ofthe theaflavin compounds include: free theaflavin, which is a compoundof the formula (II) with R₃ =H and R₄ =H; theaflavin monogallate A,which is a compound of the formula (II) with R₃₌ 3,4,5-trihydroxybenzoyl group and R₄ =H; theaflavin monogallate B, which is a compoundof the formula (II) with R₃ =H and R₄₌ 3,4,5-trihydroxy benzoyl group;and theaflavin digallate, which is a compound of the formula (II) withR₃₌ 3,4,5-trihydroxy benzoyl group and R₄₌ 3,4,5-trihydroxy benzoylgroup.

The above described tea polyphenol compounds can be prepared from tealeaves as the starting material and a method for the preparation thereofand a typical example of the product composition are described, forexample, in Japanese Patent Kokai 59-219384, 60-13780 and 61-130285 andelsewhere.

When the inventive medicament against infection with mycoplasma is to beprocessed into a medicament form or used as an additive in food, theabove described tea polyphenol as the effective ingredient or tea assuch is admixed with the base without or with dilution with water oralcohol. A preferable concentration is in the range from 0.2% to 10% fortea or about 50 μg/ml for the tea polyphenol.

The above described preventive medicament against infection withmycoplasma comprises, as the effective ingredient, a natural productwhich is drinkable and can be taken in daily life in a considerablylarge volume so that it is absolutely free from the problem ofundesirable side effects to the human body not only when it is used as amedicine, but also when it is used as an additive to food. Moreover, theeffectiveness thereof is so high that infection with mycoplasma can beeffectively inhibited by the addition thereof even in a very lowconcentration to provide a means for preventing infection withmycoplasma.

In the following, examples are given to illustrate the invention in moredetail.

EXAMPLE 1

(1) Preparation of tea and catechins:

Test solutions of tea and catechin were prepared in the followingmanner. Thus, green tea, black tea or puh-ar tea was extracted for 3hours at room temperature with a phosphate buffer solution having a pHof 7.0 in a dosage of 20 wt./vol. % followed by centrifugation at 15,000rpm for 10 minutes to give a supernatant from which test solutions wereprepared by dilution in concentrations of 20%, 4% and 0.4%. As thecatechin compound, (-)epigallocatechin gallate which exhibitedantibacterial and antitoxic activity, which is referred to as EGCghereinbelow, was selected and dissolved in the same phosphate buffersolution in concentrations of 100 μg/ml, 20 μg/ml and 2 μg/ml to givethe test solutions.

Similarly, theaflavin digallate, which is referred to as TF3hereinbelow, was dissolved in the buffer solution to give a solution of100 μg/ml concentration which was diluted 2-fold to give a solution of50 μg/ml concentration which was used as the test solution.

(2) Preparation of microbial strains and the microbial suspension:

Four strains of mycoplasmas were used for the assay including Mycoplasmapneumoniae IID 815 and IID 817 originating in human, M. salivarium IID878 isolated from human oral cavity and M. orale IID 880. Each of the M.pneumoniae strains was cultured aerobically for 5 to 6 days using theculture medium of Chanock et al. at 37° C. Each of the other strains wascultured by the GAS PAK method in the presence of carbon dioxide gas at37° C. for 4 to 5 days. After completion of the culturing, the culturebroth was centrifuged at 12,000 rpm for 50 minutes to collect themicrobial cells. The cells washed with a phosphate buffer solution wasre-dispersed in the phosphate buffer solution to give a suspension whichwas used in the assay.

(3) Assay of the mycoplasma-killing activity

Assay of the mycoplasma-killing activity was conducted in the followingmanner. Each of the diluted solutions of the teas, EGCg and TF3 wasadmixed with the same volume of the microbial suspension. Themycoplasma-added solution was diluted 10-fold to 10,000-fold and 10 μlportion of the solution, either immediately after dilution or afterincubation for 3 hours at 37° C., was added dropwise to an agar culturemedium of Chanock et al. The number of the colonies formed by culturingwas counted under a microscope of 100 times magnification and the resultwas recorded as the colony-forming units CFU/ml as a measure for theevaluation of the mycoplasma-killing activity.

As is shown in the table below, green tea and black tea exhibitedremarkable mycoplasma-killing activity against M. pneumoniae and M.orale so as to decrease the number of the microorganisms to 1/1000 orbelow even in a concentration of 0.2%. The puh-ar tea exhibitedmycoplasma-killing activity within 3 hours in a concentration of 10% or2%. On the other hand, the mycoplasma-killing activity against M.salivarium was obtained with the black tea in a concentration of 10% or2% while no mycoplasma-killing activity was exhibited against thisstrain by the green tea and puh-ar tea irrespective of theconcentration. The EGCg and TF3 exhibited remarkable mycoplasma-killingactivity in a concentration of 50 μg/ml against each of these threespecies from the moment immediately after dilution.

                                      TABLE 1                                     __________________________________________________________________________                 M. orale  M. salivarium                                                                           M. pneumoniae                                        Concen-  After 3   After 3   After 3                                          tration                                                                            As  hours of                                                                            As  hours of                                                                            As  hours of                                 Culture medium                                                                        (%)  diluted                                                                           incubation                                                                          diluted                                                                           incubation                                                                          diluted                                                                           incubation                               __________________________________________________________________________    Green tea                                                                             10   +   +     -   -     +   +                                                2    +   +     -   -     +   +                                                0.2  +   +     -   -     +   +                                        Black tea                                                                             10   +   +     +   ±  +   +                                                2    +   +     +   ±  +   +                                                0.2  +   +     -   -     +   +                                        Puh-ar tea                                                                            10   -   +     -   -     -   +                                                2    -   +     -   -     -   +                                                0.2  -   +     -   -     -   -                                        __________________________________________________________________________     + (positive): decrease in the number of microorganisms by a factor of         10.sup.2 or larger as compared with the control                               - (negative): decrease in the number of microorganisms by a factor of 10      or smaller                                                               

                  TABLE 2                                                         ______________________________________                                                         Factor of decrease                                                            in the number of micro-                                                       organisms after addition                                                      of EGCg, CFU/ml                                                          Concen-                 After 3                                               tration                 hours of                                  Culture medium                                                                            (μg/ml) As diluted   incubation*                               ______________________________________                                        EGCg added  50         <<10.sup.3   <<10.sup.3                                EGCg added  10         7.2 × 10.sup.4                                                                       <<10.sup.3                                EGCg added   1         2.8 × 10.sup.5                                                                       1.4 × 10.sup.4                      Control**   --         4.1 × 10.sup.5                                                                       3.5 × 10.sup.4                      ______________________________________                                         *: culturing time at 37° C.                                            **: phosphate buffer solution alone added                                

                  TABLE 3                                                         ______________________________________                                                         Factor of decrease                                                            in the number of micro-                                                       organisms after addition                                                      of EGCg, CFU/ml                                                          Concen-                 After 3                                               tration                 hours of                                  Culture medium                                                                            (μg/ml) As diluted   incubation*                               ______________________________________                                        EGCg added  50         4.9 × 10.sup.4                                                                       <<10.sup.3                                EGCg added  10         5.7 × 10.sup.7                                                                       8.5 × 10.sup.6                      EGCg added   1         5.7 × 10.sup.7                                                                       2.4 × 10.sup.7                      Control**   --         3.6 × 10.sup.7                                                                       3.5 × 10.sup.7                      ______________________________________                                         *: culturing time at 37° C.                                            **: phosphate buffer solution alone added                                

                  TABLE 4                                                         ______________________________________                                                         Factor of decrease                                                            in the number of micro-                                                       organisms after addition                                                      of EGCg, CFU/ml                                                          Concen-                 After 3                                               tration                 hours of                                  Culture medium                                                                            (μg/ml) As diluted   incubation*                               ______________________________________                                        EGCg added  50         3.7 × 10.sup.4                                                                       3.7 × 10.sup.4                      EGCg added  10         1.0 × 10.sup.8                                                                       1.1 × 10.sup.7                      EGCg added   1         5.8 × 10.sup.7                                                                       4.5 × 10.sup.7                      Control**   --         8.4 × 10.sup.7                                                                       4.6 × 10.sup.7                      ______________________________________                                         *: culturing time at 37° C.                                            **: phosphate buffer solution alone added                                

                  TABLE 5                                                         ______________________________________                                                              Factor of decrease in the                                            Concen-  number of microorganisms                                             tration  after addition, CFU/ml                                  Culture medium                                                                             (μg/ml)                                                                             (as diluted)                                            ______________________________________                                        TF3 added    50       3.4 × 10.sup.4                                    EGCg added   50       1.2 × 10.sup.4                                    Control**    --       1.2 × 10.sup.7                                    ______________________________________                                         **: phosphate buffer solution alone added                                

What is claimed is:
 1. A method of preventing infection in humans frommycoplasma comprising administering to said human, tea polyphenolextracted from tea in an amount sufficient to prevent infection frommycoplasma.
 2. The method of claim 1 wherein the amount administeredprovides a concentration of said tea polyphenol of about 50 μg/ml. 3.The method of claim 1 wherein said tea polyphenol is selected from thegroup consisting of epigallocatechin gallate, epicatechin gallate,epigallocatechin, epicatechin, (+)catechin and the isomer thereof, freetheaflavin, theaflavin monogallate A, theaflavin monogallate B andtheaflavin digallate.
 4. The method of claim 2 wherein said teapolyphenol is selected from the group consisting of epigallocatechingallate, epicatechin gallate, epigallocatechin, epicatechin, (+)catechinand the isomer thereof, free theaflavin, theaflavin monogallate A,theaflavin monogallate B and theaflavin digallate.
 5. The method ofclaim 9 wherein said tea polyphenol is epigallocatechin gallate.
 6. Themethod of claim 9 wherein said tea polyphenol is epicatechin gallate. 7.The method of claim 9 wherein said tea polyphenol is epigallocatechin.8. The method of claim 9 wherein said tea polyphenol is epicatechin. 9.The method of claim 9 wherein said tea polyphenol is (+)catechin and theisomer thereof.
 10. The method of claim 9 wherein said tea polyphenol isfree theaflavin.
 11. The method of claim 9 wherein said tea polyphenolis theaflavin monogallate A.
 12. The method of claim 9 wherein said teapolyphenol is theaflavin monogallate B.
 13. The method of claim 9wherein said tea polyphenol is theaflavin digallate.
 14. The method ofclaim 3, wherein the mycoplasma is selected from the group consisting ofMycoplasma pneumoniae IID 815, Mycoplasma pneumoniae IID 817, M.salivarium IID 878 and M. orale IID
 880. 15. The method of claim 3,wherein the tea polyphenol is orally administered.